The development and progression of cancer in a patient is generally found to be associated with the presence of markers in the bodily fluid of the patient, these “tumour markers” reflecting different aspects of the biology of the cancer (see Fateh-Maghadam, A. & Steilber, P. (1993) Sensible use of tumour markers. Published by Verlag GMBH, ISBN 3-926725-07-9). Tumour markers are often found to be altered forms of wild-type proteins expressed by “normal” cells, in which case the alteration may be a change in primary amino acid sequence, a change in secondary, tertiary or quaternary structure or a change in post-translational modification, for example, abnormal glycosylation. In addition, wild-type proteins which are up-regulated or over-expressed in tumour cells, possibly as a result of gene amplification or abnormal transcriptional regulation, may also be tumour markers.
Established assays for tumour markers present in bodily fluids tend to focus on the detection of tumour markers which reflect tumour bulk and as such are of value late in the disease process, for example in the diagnosis of metastatic disease. The most widely used of these markers include carcinoembryonic antigen (CEA) and the glycoprotein termed CA 15.3, both of which have been useful mainly as indicators of systemic disease burden and of relapse following therapy (Molina, R., Zanon, G., Filella, X. et al. Use of serial carcinoembryonic antigen and CA 15.3 assays in detecting relapses in breast cancer patients. (1995) Breast Cancer Res Treat 36: 41-48). These markers are of limited use earlier in the course of the disease, for example in early detection or in the screening of asymptomatic patients. Thus, in the search for tumour markers present in bodily fluid that are of use in assisting diagnosis earlier in the disease process the present inventors have sought to identify markers which do not depend on tumour bulk per se.
Differences between a wild type protein expressed by “normal” cells and a corresponding tumour marker protein may, in some instances, lead to the tumour marker protein being recognised by an individual's immune system as “non-self” and thus eliciting an immune response in that individual. This may be a humoral (i.e B cell-mediated) immune response leading to the production of autoantibodies immunologically specific to the tumour marker protein. Autoantibodies are naturally occurring antibodies directed to an antigen which an individual's immune system recognises as foreign even though that antigen actually originated in the individual. They may be present in the circulation as circulating free autoantibodies or in the form of circulating immune complexes consisting of autoantibodies bound to their target tumour marker protein.
As an alternative to the direct measurement or detection of tumour marker protein in bodily fluids, assays may be developed to measure the immune response of the individual to the presence of tumour marker protein in terms of autoantibody production. Such assays essentially constitute indirect detection of the presence of tumour marker protein. Because of the nature of the immune response, it is likely that autoantibodies can be elicited by a very small amount of circulating tumour marker protein and indirect methods which rely on detecting the immune response to tumour markers will consequently be more sensitive than methods for the direct measurement of tumour markers in bodily fluids. Assay methods based on the detection of autoantibodies may therefore be of particular value early in the disease process and possibly also in relation to screening of asymptomatic patients, for example in screening to identify individuals “at risk” of developing disease amongst a population of asymptomatic individuals. Furthermore, they may be useful for earlier detection of recurrent disease.
Tumour marker proteins observed to elicit serum autoantibodies include a particular class of mutant p53 protein, described in U.S. Pat. No. 5,652,115, which can be defined by its ability to bind to the 70 kd heat shock protein (hsp70). p53 autoantibodies can be detected in patients with a number of different benign and malignant conditions (described in U.S. Pat. No. 5,652,115) but are in each case present in only a subset of patients. For example, one study utilizing an ELISA assay for detection of autoantibodies directed against the p53 protein in the serum of breast cancer patients reported that p53 autoantibodies were produced by 26% of patients and 1.3% of control subjects (Mudenda, B., Green, J. A., Green, B. et al. The relationship between serum p53 autoantibodies and characteristics of human breast cancer, (1994) Br J Cancer 69: 4445-4449). A second tumour marker protein known to elicit serum autoantibodies is the epithelial mucin MUC1 (Hinoda, Y. et al. (1993) Immunol Lett. 35: 163-168; Kotera, Y. et al. (1994) Cancer Res. 54: 2856-2860).
WO 99/58978 describes methods for use in the detection/diagnosis of cancer which are based on evaluating the immune response of an individual to two or more distinct tumour markers. These methods generally involve contacting a sample of bodily fluid taken from the individual with a panel of two or more distinct tumour marker antigens, each derived from a separate tumour marker protein, and detecting the formation of complexes of the tumour marker antigens bound to circulating autoantibodies immunologically specific for the tumour marker proteins. The presence of such circulating autoantibodies is taken as an indication of the presence of cancer.
Cancer detection methods based on detection of circulating autoantibodies are frequently immunoassays utilizing an “immunoassay reagent” reactive with the circulating autoantibodies. Typically, the “reagents” used in such assays comprise recombinant tumour marker proteins (expressed in bacterial, insect, yeast or mammalian cells) or chemically synthesised tumour marker antigens, which may comprise substantially whole tumour marker proteins, or fragments thereof, such as short peptide antigens. Other potential sources of tumour-associated proteins for use as the basis of immunoassay reagents for the detection of anti-tumour auto-antibodies include cultured tumour cells (and the spent media used for their growth), tumour tissue, and serum from individuals with neoplasia. The majority of these sources have significant drawbacks, as discussed below.
With cultured tumour cells (and their spent media) the amount of expressed protein can vary depending on growth phase at the time of harvest, leading to variations in quality and quantity. In addition, the desired protein is generally present at low concentration, therefore it is time-consuming to purify sufficient quantities of protein. Furthermore, the cell stock will be clonal, unlike cell stock in a tumour which is likely to have become heterogeneous in nature during the growth of the neoplasm, therefore producing variations in protein (especially in the degree of glycosylation).
Recombinant proteins expressed in bacterial cells are not glycosylated, and thus significantly different from naturally glycosylated proteins. In addition, refolding of recombinantly expressed proteins may not be appropriate, thus giving an incorrect conformation for auto-antibody recognition.
Tumour tissue is usually only available in small quantities and the purification of proteins therefrom is laborious and time consuming.
Serum samples are usually available only in small quantities, therefore it is difficult to purify sufficient quantities of protein.
The present inventors have now determined that significant advantages can be gained by the use of tumour marker antigens purified from bodily fluids derived from a body cavity or space in which a tumour is present or with which it is or was associated, such as ascites fluid, pleural effusion, seroma, hydrocoele or wound drainage fluid, or from excretions, as the “reagent” in auto-antibody immunoassays. In particular, the inventors have observed that use of reagents comprising tumour marker antigens purified from bodily fluids derived from the above defined body cavities or spaces results in increased sensitivity (as compared to the use of reagents derived from a “normal” body fluid) and produces a more “clinically relevant” result. There are also significant practical advantages to be gained from the use of such fluids as a source of assay reagent.